Prognostic Significance of the Relative Load of KPC-Producing Klebsiella pneumoniae within the Intestinal Microbiota in a Prospective Cohort of Colonized Patients

ABSTRACT Increased relative bacterial load of KPC-producing Klebsiella pneumoniae (KPC-KP) within the intestinal microbiota has been associated with KPC-KP bacteremia. Prospective observational study of KPC-KP adult carriers with a hospital admission at recruitment or within the three prior months (January 2018 to February 2019). A qPCR-based assay was developed to measure the relative load of KPC-KP in rectal swabs (RLKPC, proportion of blaKPC relative to 16S rRNA gene copy number). We generated Fine-Gray competing risk and Cox regression models for survival analysis of all-site KPC-KP infection and all-cause mortality, respectively, at 90 and 30 days. The median RLKPC at baseline among 80 KPC-KP adult carriers was 0.28% (range 0.001% to 2.70%). Giannella Risk Score (GRS) was independently associated with 90-day and 30-day all-site infection (adjusted subdistribution hazard ratio [aHR] 1.23, 95% CI = 1.15 to 1.32, P < 0.001). RLKPC (adjusted hazard ratio [aHR] 1.04, 95% CI = 1.01 to 1.07, P = 0.008) and age (aHR 1.05, 95% CI = 1.01 to 1.10, P = 0.008) were independent predictors of 90-day all-cause mortality in a Cox model stratified by length of hospital stay (LOHS) ≥20 days. An adjusted Cox model for 30-day all-cause mortality, stratified by LOHS ≥14 days, included RLKPC (aHR 1.03, 95% CI = 1.00 to 1.06, P = 0.027), age (aHR 1.10, 95% CI = 1.03 to 1.18, P = 0.004), and severe KPC-KP infection (INCREMENT-CPE score >7, aHR 2.96, 95% CI = 0.97 to 9.07, P = 0.057). KPC-KP relative intestinal load was independently associated with all-cause mortality in our clinical setting, after adjusting for age and severe KPC-KP infection. Our study confirms the utility of GRS to predict infection risk in patients colonized by KPC-KP. IMPORTANCE The rapid dissemination of carbapenemase-producing Enterobacterales represents a global public health threat. Increased relative load of KPC-producing Klebsiella pneumoniae (KPC-KP) within the intestinal microbiota has been associated with an increased risk of bloodstream infection by KPC-KP. We developed a qPCR assay for quantification of the relative KPC-KP intestinal load (RLKPC) in 80 colonized patients and examined its association with subsequent all-site KPC-KP infection and all-cause mortality within 90 days. Giannella Risk Score, which predicts infection risk in colonized patients, was independently associated with the development of all-site KPC-KP infection. RLKPC was not associated with all-site KPC-KP infection, possibly reflecting the large heterogeneity in patient clinical conditions and infection types. RLKPC was an independent predictor of all-cause mortality within 90 and 30 days in our clinical setting. We hypothesize that KPC-KP load may behave as a surrogate marker for the severity of the patient’s clinical condition.

Major comments: Line 133: The number of copies of the bla-KPC gene does not necessarily mean that these were all linked to K. pneumoniae. Other Enterobacteriaceae could harbor this gene. How can you be confident that you are classifying a single species' abundance? It's unclear from the supplementary materials if an additional identification step was performed prior to PCR.
For patients who were retrospectively identified to have KPC-KP colonization, was there any verification they were still colonized on enrollment? Given that colonization can be transitory, were any subsequent cultures collected on the participants? Did you collect any information on other outcome metrics, such as total infections? If, as you propose, KPC-KP colonization signifies dysbiosis, bacterial translocation could occur from any species, not just KP (see: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193040/). This would be especially interesting to know given the relatively low loads of KPCs in your study.
Line 362-365: Why does colonization increase mortality, independent of infection? I'm not sure I find the argument that it represents a decompensation of underlying disease cogent.
Minor comments: The manuscript would benefit from spellcheck.
Line 83: Do you mean surrogate instead of subrogate?
Line 112: How is endemicity defined? Is this based on colonization screening or isolates from clinical infections?
Line 115: When you say "first diagnosis" do you mean the individuals may have been colonized for an indefinite period of time? How were the patients selected (beyond colonization status)? Was it just a convenience sample of the first 80 patients identified? Where in the hospital were they located (surgical, medical, all wards)? Is Day 0 the first day of admission, the first day with a positive swab, or the start of the study (if it was cross-sectional)? It would be helpful to know more about the study design for the purposes of generalizability.
Why did you stratify 30-day mortality by LOH >14 days and 90-day mortality by LOH >20 days? Were there clear splines in the data?
Hospitalization at recruitment may not be a significant factor in your analysis because of the small number of people who were not hospitalized. I would include this as a limitation.
I'm surprised that previous hospitalization was not considered in the list of risk factors. Patients who are high utilizers of healthcare might be expected to have higher mortality in general, but this was not accounted for in the analysis. Authors studied bacterial load of KPC producing Klebsiella pneumoniae within the intestinal microbiota in a prospective cohort of colonized patients. They also analyze the prognosis of this value. Bacteria load unlike other studies did not influence in latter infection. A qPCR assay was used for the study of bacterial load. The statistical analysis is sound General comments The inclusion criteria were patients with a first diagnosis of intestinal KPC-KP colonization during a hospital admission at the time of recruitment or within the three prior months. It seems different groups for the analysis. Do the authors perform a separate analysis in both groups? How do the authors control the sampling (rectal swabs and not perirectal swabs?) Include type and/or trade mark of rectal swabs. This group has experience with intestinal decontamination (reference 34). Was the use of this procedure considered an exclusion criteria? The total number of patients is very low and this might have bias the results. Nevertheless the did a rigorous analysis of the data obtained. Specific comments Title. I will recommend to use the acronysms KPC instead "Klebsiella pneumoniae carbapenemase" This will make the title more readable. Line 54. Is this a specific 16S rRNA for K. pneumoniae or total 16S rRNA? Line 84. Immunological conditions? This might be out of the scope Line 193. Range of patient's age is very high. Were younger patients excluded? Line 223. Antibiotic susceptibility instead antibiotic sensitivity Line 227. There is no EUCAST criteria for tigecycline and K. pneumoniae. Do the authors used the PK-PD breakpoint for this antibiotic? Line 236. Was the antibiotic treatment influence this figure? Line 363-365. This can be due to the progressive elimination of Firmicutes and increase of Proteobacteria with aging Table S3. Include S / I / R in this order.

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Microbiology Spectrum
Review of "Prognostic significance of the relative load of Klebsiella pneumoniae carbapenemaseproducing Klebsiella pneumoniae within the intestinal microbiota in a prospective cohort of colonized patients."

Summary:
The submitted article provides a unique look at the significance of bacterial load of KPC-producing Klebsiella pneumoniae in predicting subsequent infection or death. The authors developed a PCR method to provide a relative value of bla-KPC genes. While the bacterial load did not correlate with infection, it was independently correlated with death. The Giannella Risk Score was associated with subsequent infection.
Major comments: Line 133: The number of copies of the bla-KPC gene does not necessarily mean that these were all linked to K. pneumoniae. Other Enterobacteriaceae could harbor this gene. How can you be confident that you are classifying a single species' abundance? It's unclear from the supplementary materials if an additional identification step was performed prior to PCR.
For patients who were retrospectively identified to have KPC-KP colonization, was there any verification they were still colonized on enrollment? Given that colonization can be transitory, were any subsequent cultures collected on the participants? Did you collect any information on other outcome metrics, such as total infections? If, as you propose, KPC-KP colonization signifies dysbiosis, bacterial translocation could occur from any species, not just KP (see: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193040/). This would be especially interesting to know given the relatively low loads of KPCs in your study.
Line 362-365: Why does colonization increase mortality, independent of infection? I'm not sure I find the argument that it represents a decompensation of underlying disease cogent.
Minor comments: The manuscript would benefit from spellcheck.
Line 83: Do you mean surrogate instead of subrogate?
Line 112: How is endemicity defined? Is this based on colonization screening or isolates from clinical infections?
Line 115: When you say "first diagnosis" do you mean the individuals may have been colonized for an indefinite period of time? How were the patients selected (beyond colonization status)? Was it just a convenience sample of the first 80 patients identified? Where in the hospital were they located (surgical, medical, all wards)? Is Day 0 the first day of admission, the first day with a positive swab, or the start of the study (if it was cross-sectional)? It would be helpful to know more about the study design for the purposes of generalizability.
Why did you stratify 30-day mortality by LOH >14 days and 90-day mortality by LOH >20 days? Were there clear splines in the data?
Hospitalization at recruitment may not be a significant factor in your analysis because of the small number of people who were not hospitalized. I would include this as a limitation.
I'm surprised that previous hospitalization was not considered in the list of risk factors. Patients who are high utilizers of healthcare might be expected to have higher mortality in general, but this was not accounted for in the analysis. Response to reviewer comments for the article entitled "Prognostic significance of the relative load of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae within the intestinal microbiota in a prospective cohort of colonized patients" (Spectrum02728-21)" to be considered for publication in Microbiology Spectrum as "Original Article".

Summary:
The submitted article provides a unique look at the significance of bacterial load of KPCproducing Klebsiella pneumoniae in predicting subsequent infection or death. The authors developed a PCR method to provide a relative value of bla-KPC genes. While the bacterial load did not correlate with infection, it was independently correlated with death. The Giannella Risk Score was associated with subsequent infection.

Major comments:
Major comment 1: Line 133: The number of copies of the bla-KPC gene does not necessarily mean that these were all linked to K. pneumoniae. Other Enterobacteriaceae could harbour this gene. How can you be confident that you are classifying a single species' abundance? It's unclear from the supplementary materials if an additional identification step was performed prior to PCR.
Response: For all rectal swabs, quantification of bacterial load was performed by two methods in parallel: qPCR and culture on selective growth medium. This was followed by identification of any Enterobacterales growing in CRE-selective culture medium, following standard microbiological procedures. We have re-written the main Methods section to make this clearer. In addition, we have included a new Supplementary Figure S1 (also included below) summarizing the processing of rectal swabs in our study, which highlights all molecular and microbiological tests performed for each sample.

Spectrum02728-21R1_Response to Reviewers
Major comment 2: For patients who were retrospectively identified to have KPC-KP colonization, was there any verification they were still colonized on enrolment? Given that colonization can be transitory, were any subsequent cultures collected on the participants?

Response:
Regarding the first question, yes, following signing of the informed consent by patients who were retrospectively identified, a rectal swab sample was obtained for confirmation of colonization persistence, considered as day 0 of follow-up. Among these patients, only those with a positive KPC-KP culture on day 0 were included in the study, while uncolonized patients were not included in our cohort.
Regarding the second question, the answer is yes. In this prospective cohort study, all patients were cited for monthly visits for clinical and microbiological follow-up. In the case of patients with reduced mobility discharged to a residential care or nursing home, a dedicated nurse travelled to the patient´s residence to perform the monthly visits during the study period. In the present paper we focus on the impact of baseline KPC-KP relative intestinal load (day 0) on infection and mortality within 3 months of enrolment. Due to space limitations, results on the natural history of KPC-KP colonization, as well as progression of KPC-KP bacterial load kinetics and risk factors associated with eradication of KPC-KP in our cohort study are the focus of a different analysis and paper.
All this information has been clarified in Methods, as follows: Lines 117-148 (Marked copy): The study recruitment period was from January 2018 to February 2019. Inclusion criteria were adult patients with a first KPC-KPpositive rectal surveillance culture during a hospital admission throughout the recruitment period or within the three prior months (November-December 2018). During the study period, KPC-KP rectal colonization screening was systematically performed in our hospital by means of a rectal swab culture in patients admitted to high-risk units (intensive care unit and hematology unit) and those undergoing abdominal surgery or transplants. In addition, a colonization study may have been requested by the clinician for various reasons: previous admission to high-risk units, origin of the patient from a healthcare center, or sharing a room with colonized or infected patients. Subjects with a first KPC-KP colonization during a hospital stay in the three months prior to start of recruitment were identified through review of hospital Spectrum02728-21R1_Response to Reviewers records, contacted by phone and invited to participate in the study. Following the signing of the informed consent, a rectal swab sample was obtained for confirmation of KPC-KP colonization and quantification of KPC-KP intestinal relative load (baseline RL KPC ) in all patients and this was considered the start date of follow-up (day 0). Only those patients with a positive KPC-KP culture on day 0 of follow-up were included in the study, while uncolonized patients were excluded. In addition, colonized patients who received a clinical indication of selective intestinal decolonization with oral nonadsorbable antibiotics during follow-up were excluded from the present analysis and they are described elsewhere (23). All patients were followed until 90 days or death. Patients were seen at the hospital on a monthly basis for clinical and microbiological follow-up by means of rectal swab screening. In the case of patients with reduced mobility, a dedicated nurse went to the patient's residence for clinical and microbiological follow-up visits.
Major comment 3: Did you collect any information on other outcome metrics, such as total infections? If, as you propose, KPC-KP colonization signifies dysbiosis, bacterial translocation could occur from any species, not just KP (see: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193040/). This would be especially interesting to know given the relatively low loads of KPCs in your study.
Response: This is an interesting hypothesis, which unfortunately was not studied in our work. Our study focused on KPC-KP infections. Unfortunately, data on the total number of infections was not recorded during data collection. As the reviewer suggests, during a state of dysbiosis, niches left open after microbiota alteration may be occupied by overgrowth of microorganisms that have pathogenic potential at high densities. Members of the Proteobacteria, such as Escherichia coli or K. pneumoniae, normally represent < 2% of the microbiota, but in a dysbiotic state these bacteria can represent up to 30% of the total species. On the other hand, with the healthcare personnel acting as a transmission vector, this ecological gap may be filled by other environmental opportunistic pathogens that frequently carry genetic determinants of resistance in the hospital setting. It would be of interest to analyze this hypothesis in future studies in our hospital.

Major comment 4:
Line 362-365: Why does colonization increase mortality, independent of infection? I'm not sure I find the argument that it represents a decompensation of underlying disease cogent.

Response:
We agree with the reviewer. To keep the word limit, we oversimplified our discussion of this point. We have re-written this part of the discussion considering major comments 3 and 4, as follows: Before: We hypothesize that the relative intestinal load of KPC-KP may behave as a surrogate marker for decompensation of the underlying disease, comorbidities or patient's immune status (immunosenescence) in our elderly patient population, which ultimately may lead to patient´s death. Now (Lines 411-428, marked copy): Age-related alterations in the gut microbiome are influenced by factors such as progressive physiological deterioration, and lifestylelinked factors including diet, medication and reduced social contact (38). In people over the age of 70, a study reported a decrease in anaerobic bacteria such as Bifidobacterium spp., which has a role in in the stimulation of the immune system and metabolic processes, and an increase in Clostridium and Proteobacteria (39). Hospitalization may exacerbate microbiota dysbiosis in frail, older people as a result of Spectrum02728-21R1_Response to Reviewers medication and exposure to healthcare invasive procedures. The niches left open within the intestinal microbiota may be occupied by overgrowth of microorganisms that have pathogenic potential at high densities, with the healthcare personnel acting as a transmission vector in the hospital environment. Members of the Proteobacteria, such as Escherichia coli or K. pneumoniae, normally represent less than 2% of the microbiota, but in a dysbiotic state these bacteria can represent up to 30% of the total species (18). We hypothesize that an increased intestinal load of KPC-KP within the gut microbiota in our clinical context may behave as a surrogate marker of debilitated health condition because of frailty, higher burden of comorbidities, or age-related alterations in the gut microbiome and immune dysfunction (immune senescence), which may in turn lead to higher risk of infection or death.

Minor comments:
Minor comment 1: The manuscript would benefit from spellcheck.
Response: Thank you. We have reviewed the manuscript thoroughly to correct any spelling errors.
Minor comment 2: Line 83: Do you mean surrogate instead of subrogate?
Response: Corrected, thank you.
Minor comment 3: Line 112: How is endemicity defined? Is this based on colonization screening or isolates from clinical infections?
Response: After controlling an initial outbreak by a KPC3-KP ST512 clone in the summer of 2012, endemicity was reached in our hospital and has prevailed to date, with cases of colonisation and infection with no epidemiological relationship between them (and therefore without epidemiological outbreak criteria). In high-risk units (ICU, haematology) this criterion is based on colonisation screening. In the rest of the hospital units, it is based on isolations of clinical infections. This general information has been included in Methods, "Study design" section (Lines 113-148, marked copy).
Minor comment 4: Line 115: When you say "first diagnosis" do you mean the individuals may have been colonized for an indefinite period of time? How were the patients selected (beyond colonization status)? Was it just a convenience sample of the first 80 patients identified? Where in the hospital were they located (surgical, medical, all wards)? Is Day 0 the first day of admission, the first day with a positive swab, or the start of the study (if it was crosssectional)? It would be helpful to know more about the study design for the purposes of generalizability.

Response:
We thank the reviewer for highlighting the need to expand and clarify the information on study design in the main manuscript. As explained in our response to "Major comment 2", we have rewritten the Methods section accordingly. Thus, details regarding study design, selection of patients, and criteria for rectal swab surveillance in our hospital during the study period are now explained in detail in Methods, "Study design" section (Lines 113-148, marked copy).
Minor comment 5: Why did you stratify 30-day mortality by LOH >14 days and 90-day mortality by LOH >20 days? Were there clear splines in the data?
Response: Dichotomization of the variable length of hospital stay (LOHS) was performed according to ROC curve analyses. This explanation has been included in the text (Lines 319-320, marked copy), and the ROC curves are shown in Supplementary Figure S9.
Minor comment 6: Hospitalization at recruitment may not be a significant factor in your analysis because of the small number of people who were not hospitalized. I would include this as a limitation.
I'm surprised that previous hospitalization was not considered in the list of risk factors. Patients who are high utilizers of healthcare might be expected to have higher mortality in general, but this was not accounted for in the analysis.
Response: Thank you. This variable was included in our analysis as "Hospitalization in the previous three months" (please, see Table 1). We explored its impact on infection and mortality but found no independent association in our multivariable models. Moreover, in our work, we conducted a first exploratory analysis based on survival Random Forest to identify variables with a strong relative importance for survival. This analysis was previously not mentioned in the manuscript´s methods due to space limitations, however we consider it of interest for the reader and have now included a brief description in Statistical Methods (Lines 226-231, marked copy). In addition, we have also included a new Supplementary Figure S2, which summarizes the results of this random survival forest analysis. As shown in this Figure  S2, the variable "Hospitalization in the previous three months" showed negative variable importance with regards to the outcome. Table 7: There are two columns listed as multivariable analysis. Presumably the first one is univariable analysis.